As set forth in "Electrical Wiring of Redox Enzymes," Heller, Acc. Chem. Res. Vol. 23, No. 5, 1990, in many enzymes, the redox centers thereof are located too remote from the outermost protein surface to be electrically accessible for purposes of providing direct electrical interaction between the enzyme and its associated electrode. Accordingly, it was difficult to promote chemical reactions by directly interacting the electrode and enzyme and it was similarly difficult to monitor chemical reactions involving the enzyme directly at the electrode. Due to the inaccessible nature of the redox centers of these enzymes, so-called mediators or electron shuttles have been physically admixed with the enzyme in a membrane or in the test system, or, in certain cases, the mediators or shuttles were chemically bonded to the enzyme to enhance electron transfer from a reactant or desired analyte through the enzyme to the electrode.
For example, mediated glucose sensors involving water soluble electron acceptors, such as ferricyanide, quinones, and various organic dyes have been tried. In European Patent Publication 0 278 647, modification of flavo protein enzymes such as glucose oxidase and D-amino acid oxidase through chemical attachment of electronically active materials thereto such as a ferroceneamide or a ferroceneacetamide is disclosed. Additionally, as reported in this publication, conversion of functional moieties on the enzymes, such as conversion of enzyme tyrosine-amides to enzyme dihydroxyphenylalanine amides, provides improvement in direct electrical interaction between enzyme and electrode.
In U.S. Pat. No. 4,840,893 (Hill et al.), a host of mediator enzyme systems is disclosed. Disclosed mediators include ruthenium complexes, ferrocenes, carbon-boron compounds, phenazine dyes, and metalloporphyrins. Enzyme/substrate pairs, whose behavior in association with mediator compounds has been purportedly studied, include flavo-proteins, PQQ enzymes, cytochrome b-linked enzymes, and metallo flavo proteins.
U.S. Pat. No. 5,089,112 teaches artificial redox compounds (i.e., mediators) that are covalently bound to a flexible polymer backbone. Disclosed polymers include siloxanes, polyphosphazene, poly(ethylene oxide) and poly(propylene oxide).
Redox-conducting epoxy cements, including conductive polyvinylpyridine osmium bipyridine polymers for use in chemically bonding with flavo protein enzymes, specifically glucose oxidase, are disclosed by Heller in the aforenoted Accounts of Chemical Research article and in "Redox Polymer Films Containing Enzymes. 1 A Redox-Conducting Epoxy Cement: Synthesis, Characterization, and Electrocatalytic Oxidation of Hydroquinone," J. Phys. Chem. 1991, 95, pp. 5970-5975.
Despite the efforts in the art, there remains a need to provide a conductive polymer that will chemically link (i.e., bond) to a PQQ based dehydrogenase enzyme to serve as an electron mediator or shuttle system so that direct electron transfer can be made from a reactant or analyte in contact with the enzyme to an electrode surface in contact with the enzyme-polymer mass.
More specifically, in the food and beverage industry, there is a need to provide for accurate determination of D-fructose concentration in liquids wherein an enzyme electrode containing PQQ based membrane bound fructose dehydrogenase enzyme is used in an amperometric measurement systems. Of further importance is the provision of a PQQ based membrane bound fructose dehydrogenase enzyme that is chemically bound to a water soluble, conductive polymer is contact with an electrode to provide for direct amperometric measurement without the requirement of additional mediator or electron shuttle moieties added to the reaction system.